Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression

ABSTRACT

Transgenic animals containing a nucleic acid sequence encoding TCL1 operably linked to transcriptional control sequences directing expression to B cells are described. Such transgenic animals provide a useful animal model system for human B cell chronic lymphocytic leukemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/427,629, filed Apr. 29, 2003, abandoned, which claims priority ofU.S. Provisional Application No. 60/376,464, filed on Apr. 29, 2002. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The present invention was supported by the U.S. National CancerInstitute under Grant Nos. PO1-CA76259 and PO1-CA81534. The governmentmay have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to transgenic animal models forlymphoproliferative disorders. More particularly, the present inventionrelates to transgenic animal models for human B cell chronic lymphocyticleukemia. The present invention also relates to methods of using animalmodels for testing modalities of treating and preventinglymphoproliferative disorders.

BACKGROUND OF THE INVENTION

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemiain the Western world with as many as 10,000 new cases reported each yearin the United States (Rai, K. & Patel, D. C. (1995) in Hematology: BasicPrinciples and Practice, eds. Hoffman et al. (Churchill Livingstone, NewYork), pp. 1308-1321; Landis, S. H., Murray, T., Bolden, S. & Wingo, P.A. (1998) CA cancer J. Clin. 48, 6-29). Characteristically B-CLL is adisease of elderly people resulting from the progressive accumulation ofa leukemic clone that may be derived from a normal CD5+ B lymphocyte(Caligaris-Cappio, F., Gobbi, M., Bofill, M. & Janossy, G. (1982) J.Exp. Med. 155, 623-628). B-CLL has a consistent association with CD5expression (Caligaris-Cappio, F., Gobbi, M., Bofill, M. & Janossy, G.(1982) J. Exp. Med. 155, 623-628) and while there is still a debate onthe role and significance of CD5 expression on B cells, it remainsreasonable to consider CD5+ B cells as the normal counterpart of B-CLL(Boumsell, L., Bernard, A.; Lepage, V., Degos, L., Lemerle, J. &Dausset, J., L. (1978) Eur. J. Immunol. 8, 900-904; Kantor, A. B. (1991)Immunol. Today 12, 389-391).

Human hematopoietic malignancies are often caused by chromosometranslocations involving either T-cell receptor (TCR) or Immunoglobulin(Ig) loci (Croce, C. M. (1987) Cell 49, 155-156). These chromosomebreakpoints juxtapose enhancer elements of TCR and Ig loci toproto-oncogenes, leading to tumor initiation through oncogenederegulation (Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D.,Gallo, R. C. & Croce, C. M. (1982) Proc. Natl. Acad. Sci. USA 79,7824-7827; ar-Rushdi, A., Nishikura, K., Erikson, R. W., Rovera, G. &Croce C. M. (1983) Science 222, 390-393).

The TCL1 gene, which has been identified at chromosome 14q32.1(Virgilio, L., Narducci, M. G., Isobe, M., Billips, L. G., Cooper, M.D., Croce, C. M. & Russo, G. (1994) Proc. Natl. Acad. Sci. USA 91,12530-12534), is commonly activated by inversions or translocations thatjuxtapose it to a T cell receptor locus at 14q11 or 7q35. The TCL1 geneis involved in chromosomal translocations and inversions in matureT-cell leukemias. These leukemias are classified either as Tprolymphocytic leukemias, which occur very late in life, or as T chroniclymphocytic leukemias, which often arise in patients with ataxiatelengiectasia (AT) at a young age. TCL1 has been found to be overexpressed in sporadic and ataxia telangiectasia associated T-PLL(Narducci, M. G., Stoppacciaro A., Imada, K., Uchiyama, T., Virgilio,L., Lazzeri, C., Croce, C. M. & Russo G. (1997) Cancer Res. 57,5452-5456; Thick, J., Metacalfe, J. A., Mak, Y-F., Beatty, D.,Minegishi, Dyer, M. J. S., Lucas, G. & Taylor, A. M. R. (1996) Oncogene12, 379-386). In transgenic animals the deregulated expression of TCL1leads to mature T-cell leukemia, demonstrating the role of TCL1 in theinitiation of malignant transformation in T-cell neoplasia. Evidence hasbeen provided that TCL1 is a bona fide oncogene; a transgenic mousemodel has been developed in which ectopic expression driven by the lckpromoter in the T-cell compartment results in the development of matureT-cell leukemias after a long latency period, in a pattern closelyresembling human mature T-cell leukemia (Virgilio, L., Lazzeri, C.,Bichi, R., Nibu, K., Narducci, M. G., Russo G., Rothstein, J. L. & CroceC. M. (1998) Proc. Natl. Acad. Sci. USA 95, 3885-3889). In normalT-cells, TCL1 is only expressed at the very early CD4−/CD8− doublenegative stage, whereas more mature T-cells lack TCL1 expression(Virgilio, L., Narducci, M. G., Isobe, M., Billips, L. G., Cooper, M.D., Croce, C. M. & Russo, G. (1994) Proc. Natl. Acad. Sci. USA 91,12530-12534). In the B-cell lineage, the product of the TCL1 gene, Tcl1,has been found in pre-B-cells, surface IgM expressing virgin B-cells,mantle cells and germinal center B-cells, whereas it is down-regulatedat later stages of B-cell differentiation, i.e. plasma cells (Virgilio,L., Narducci, M. G., Isobe, M., Billips, L. G., Cooper, M. D., Croce, C.M. & Russo, G. (1994) Proc. Natl. Acad. Sci. USA 91, 12530-12534).Interestingly, high levels of Tcl1 have been found in a broad variety ofhuman tumor-derived B-cell lines ranging from pre-B cell to mature Bcell and in many cases of B-cell neoplasias (Takizawa, J., Suzuki, R.,Kuroda, H., Utsunomiya, A., Kagami, Y., Joh, T., Aizawa, Y., Ueda, R. &Seto, M. (1998) Jpn. J. Cancer Res. 89, 712-718; Narducci, M. G.,Pescarmona, E., Lazzeri, C., Signoretti, S., Lavinia, A. M., Remotti,D., Scala, E., Baroni, C. D., Stoppacciaro, A., Croce, C. M., et al.(2000) Cancer Res. 60, 2095-2100). To further elucidate the role of TCL1in B cell development and in B cell neoplasia, the present inventorgenerated transgenic mice under the control of a promoter and enhancerwhose activity specifically targets expression of the transgene to theB-cell compartment (Shaw, A. C., Swat, W., Ferrini, R., Davidson, L. &Alt, F. W. (1999) J. Exp. Med. 189, 123-129). It is demonstrated hereinthat Eμ-TCL1 transgenic mice develop a disease resembling human CLL. Themice develop at first a preleukemic state evident in blood, spleen, bonemarrow, peritoneal cavity and peripheral lymphoid tissue, developinglater a frank leukemia with all the characteristics of CLL. Thesefindings strongly indicate that TCL1 and/or other gene(s) in the TCL1pathway are responsible for the initiation of human CLL. The animalmodel described herein thus provides an in vivo model for human B-CLL.

SUMMARY OF THE INVENTION

The present invention is directed, in part, to novel animal models forlymphoproliferative disorders. Specifically, according to an aspect ofthe invention, there are provided animal models for human B-CLL.

In one embodiment of the invention, a transgenic animal is providedwhose genome comprises a nucleic acid construct or transgene comprisingat least one transcriptional regulatory sequence capable of directingexpression to B cells operably linked to a nucleic acid sequenceencoding TCL1. In a preferred embodiment of the invention, the transgenecomprises a DNA sequence encoding TCL1 which has been placed under thetranscriptional control of a V_(H) promoter and a Ig_(H)-μ enhancer.

In another embodiment of the invention, white blood cells from atransgenic animal exhibiting lymphoproliferation may be transferred to asecond animal (which may be a non-transgenic animal), thereby inducing arapid onset of lymphoproliferative disease in the second “recipient”animal.

According to another embodiment of the invention, potential therapeuticmodalities for preventing and/or treating lymphoproliferative disordersmay be tested by measuring the anti-lymphoproliferative activity of suchmodalities in animals produced according to one or more aspects of theinvention.

These as well as other important aspects of the invention will becomemore apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawings willbe provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1D illustrate the production of Eμ-TCL1 transgenic mice. (a)Schematic representation of the construct used to generate the mice.Restriction sites: X, XhoI; S, SalI; E, EcoRV; B, BssHII. (b) Southernblot analysis of DNA isolated from the tails of the first transgenicprogeny for both founders and non-TG control. (c) Immunoblot analysis onprotein extracts from transgenic (F3 and F10) and non-transgenic(C=control) mouse tissues. 697=pre-B leukemic cell line 697 whichexpresses high levels of Tcl1 protein (9). (d) TCL1 expression on gatesubsets of splenic B cells. The upper panel refers to the F3 progeny,the lower panel shows the F10 progeny (Blue=transgenic (TG),Red=non-transgenic (Non-TG)).

FIGS. 2A-2E show the characterization of Eμ-TCL1 mice. (a) Correlationbetween IgM and CD5 expression in single cell suspensions from bonemarrow, spleen and peritoneal cavity in transgenic animals and a non-TGlittermate. (b) Hematoxylin and eosin-stained spleen of mouse showing anexpanded marginal zone (MZ) in Eμ-TCL1 animals. (c) Immunodetection ofTcl1 protein in lymphoid cells of the MZ. (d) Cell cycle analysis on IgMand CD5 subsets of cells by PI-labeling. (e) Cell proliferation analysisby BrdU incorporation.

FIGS. 3A-3B show the analyses of IgH gene configuration. (a) IgH generearrangements were analyzed by Southern blot on EcoRI and StuI-digestedsplenocyte DNAs. Transgenic mice (+) of 7, 8 and 9 months showrearranged bands (asterisks). No predominant rearrangement is observedin the youngest mice. Controls (−) are non-TG mice with the genomic6.5-kb EcoRI and 4.7-kb StuI fragments. (b) Southern blots on DNAisolated from bone marrow, spleen and peritoneal cavity of transgenicmice (#40, #41) with the CD5+/IgM+ expanded population. IgH genepredominant rearrangements were detected in spleen and peritoneal cavity(asterisks). DNA from spleen of non-TG mouse was used as control.

FIGS. 4A-4H show histopathological analyses of the Eμ-TCL1 mice. (a)Blood smear stained with Wright Giemsa showing an increased number ofcirculating lymphocytes. (b) High magnification of the blood smear. (c)Histology of spleen, liver (e) and cervical lymph node (g) afterhematoxylin-eosin staining. (d) Immunodetection of Tcl1 protein inspleen, liver (f) and cervical lymph node (h). Insets: negative controlsin which the primary antibody has been omitted.

FIG. 5 shows the Southern blot analysis of IgH gene rearrangements inleukemias from transgenic mice. DNAs from leukemic mice and a littermatecontrol were digested with Stu I. The strong 4.7-kb bands represent thegene in its germline configuration. Clonal rearrangements are indicatedby asterisks. Lanes 1 and 2=leukemic mice from transgenic line F3; lanes3 and 4=leukemic mice from transgenic line F10; Lane 5=non-transgenicmouse.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

The term “animal” is used herein to include all vertebrate animals,except humans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. A “transgenic animal”is an animal containing one or more cells bearing genetic informationreceived, directly or indirectly, by deliberate genetic manipulation ata subcellular level, such as by microinjection or infection withrecombinant virus. This introduced nucleic acid molecule may beintegrated within a chromosome, or it may be extra-chromosomallyreplicating DNA. Transgenic animals may include, but are not limited to,those animals in which the genetic information was introduced into agerm line cell, thereby conferring the ability to transfer theinformation to offspring. If such offspring in fact possess some or allof that information, then they, too, are transgenic animals.

“Anti-lymphoproliferative activity” refers to any activity whichinhibits, prevents, and/or destroys the growth of any neoplasmsassociated with a lymphoproliferative disorder.

As used herein, the term “expanded population of CD5+ B cells” refers toa population of B cells in an experimental animal that represents anincrease in the number of CD5+ B cells and/or the proportion of CD5+ Bcells relative to other subtypes of B cells, as compared to that of acontrol animal, as demonstrated, for example, herein in Example 2.

The term “enhancer” is used according to its art-recognized meaning. Itis intended to mean a sequence found in eukaryotes and certaineukaryotic viruses which can increase transcription from a gene whenlocated (in either orientation) up to several kilobases from the genebeing studied. These sequences usually act as enhancers when on the 5′side (upstream) of the gene in question. However, some enhancers areactive when placed on the 3′ side (downstream) of the gene. In somecases, enhancer elements can activate transcription from a gene with no(known) promoter.

“Lymphoproliferative” refers to that which pertains to or ischaracterized by proliferation of the cells of the lymphoreticularsystem; the term is generally used to refer to a group of malignantneoplasms. “Lymphoreticular” refers to the cells or tissues of both thelymphoid and reticuloendothelial systems. “Lymphoproliferative disorder”(or “lymphoproliferative disease” or “lymphoproliferative condition”)refers to one of a group of malignant neoplasms arising from cellsrelated to the common multipotential, primitive lymphoreticular cellthat includes, among others, the lymphocytic, histiocytic, and monocyticleukemias, multiple myeloma, plasmacytoma, Hodgkin's disease, alllymphocytic lymphomas, and immunosecretory disorders associated withmonoclonal gammopathy. As used herein, “lymphoproliferative disorder”,“lymphoproliferative disease” or “lymphoproliferative condition” mayalso refer to a physiological state in which the proliferation,multiplication and/or accumulation of cells of the lymphoreticularsystem is altered relative to a normal or control animal, but theaffected animal does not yet necessarily exhibit symptoms of one of theneoplasms described above. As used herein, a “preleukemic” state refersto such a lymphoproliferative condition which precedes the developmentof overt symptoms of leukemia.

“Neoplasia” refers to the formation of a neoplasm, i.e., the progressivemultiplication of cells under conditions that generally would notelicit, or would likely cause cessation of, multiplication of normalcells.

The term “promoter” is used according to its art-recognized meaning. Itis intended to mean the DNA region, usually upstream to the codingsequence of a gene or operon, which binds RNA polymerase and directs theenzyme to the correct transcriptional start site.

“Therapeutic modality” refers to any means of treating and/or preventinga given disease, condition or disorder.

The term “transcriptional regulatory sequence” is used according to itsart-recognized meaning. It is intended to mean any DNA sequence whichcan, by virtue of its sequence, cause the linked gene to be either up-or down-regulated in a particular cell. In the case of a promoter, thepromoter will generally be adjacent to the coding region. In the case ofan enhancer, however, an enhancer may function at some distance from thecoding region such that there is an intervening DNA sequence between theenhancer and the coding region.

“Transgene” refers to a nucleic acid sequence introduced into one ormore cells of a non-human animal by way of human intervention such as byway of the methods described below.

The introduced genetic information may be foreign to the species ofanimal to which the recipient belongs, foreign only to the particularindividual recipient, or genetic information already possessed by therecipient. In the last case, the introduced genetic information may bedifferently expressed compared to the native endogenous gene.

To direct expression of the genetic information, which may include a DNAsequence encoding a particular protein (or “coding region”), the codingregion of interest may be coupled to at least one transcriptionalregulatory sequence in a functional manner. Transcriptional regulatorysequences may be used to increase, decrease, regulate or designate tocertain tissues or to certain stages of development the expression of agene. The transcriptional regulatory sequences need not be naturallyoccurring sequences.

To produce transgenic animals, any method known in the art forintroducing a recombinant construct or transgene into an embryo, suchas, for example, microinjection, cell gun, transfection, liposomefusion, electroporation, and the like, may be used. However, the mostwidely used method for producing transgenic animals, and the methodpreferred according to the present invention, is microinjection, whichinvolves injecting a DNA molecule into the male pronucleus of fertilizedeggs (Brinster et al, 1981; Costantini et al, 1981; Harbers et al, 1981;Wagner et al, 1981; Gordon et al, 1976; Stewart et al, 1982; Palmiter etal, 1983; Hogan et al, 1986; U.S. Pat. Nos. 4,870,009; 5,550,316;4,736,866; 4,873,191). The above methods for introducing a recombinantconstruct/transgene into mammals and their germ cells were originallydeveloped in the mouse. These methods were subsequently adopted for usewith larger animals, including livestock species (WO 88/00239, WO90/05188, WO 92/11757; and Simon et al, 1988). Microinjection of DNAinto the cytoplasm of a zygote can also be used to produce transgenicanimals.

The present invention is not limited to any one species of animal, butprovides for any appropriate non-human vertebrate species. For example,while mouse is a preferred vertebrate species for producing transgenicanimals, other non-limiting examples including guinea pigs, rabbits,pigs, sheep, etc., may be suitably used. The success rate for producingtransgenic animals by microinjection is highest in mice, whereapproximately 25% of fertilized mouse eggs into which the DNA has beeninjected, and which have been implanted in a female, will develop intotransgenic mice. A lower success rate has been achieved with rabbits,pigs, sheep and cattle (Jaenisch, 1988; Hammer et al, 1985 and 1986;Wagner et al, 1984).

A nucleic acid molecule is said to be “capable of expressing” or“capable of directing expression of” a protein if it contains nucleotidesequences which contain transcriptional and translational regulatoryinformation, and such sequences are “operably linked” to nucleotidesequences which encode the protein. An operable linkage is a linkage inwhich regulatory nucleic acid sequences and the nucleic acid sequencesought to be expressed are connected in such a way as to permit geneexpression. The regulatory regions needed for gene expression in generalmay include, for example, transcriptional regulatory sequences such as,for example, a promoter region, as well as DNA sequences which, whentranscribed into RNA, will signal the initiation of protein synthesis.Such regions will normally include those 5′-non-coding sequencesinvolved with initiation of transcription and translation. A promoterregion would be operably linked to a DNA sequence if the promoter werecapable of effecting transcription of that DNA sequence. Thus, in oneembodiment of the present invention, a sequence encoding TCL1 isoperably linked to transcriptional regulatory sequences directingexpression to B cells, to generate a recombinant construct or“transgene” that is then introduced into a fertilized egg.

The methods for evaluating the presence of the introduced transgene aswell as its expression are readily available and well-known in the art.Such methods include, but are not limited to, DNA (Southern)hybridization to detect the exogenous DNA, polymerase chain reaction(PCR), polyacrylamide gel electrophoresis (PAGE) and blots to detectDNA, RNA or protein.

B-CLL is the most common leukemia in humans and its pathogenesis isstill unknown. Transgenic mice in which the expression of TCL1 wastargeted to B cells develop a lymphoproliferative disease closelyresembling human CLL. The results provided herein strongly indicate thatTCL1 and/or other gene(s) in the TCL1 pathway are responsible for theinitiation of human B-CLL. Herein is provided, according to an aspect ofthe invention, an animal model which may be used to investigate themechanisms underlying the initiation and progression of human B-CLL.This animal model may also be used in the development and testing ofnovel therapeutic modalities useful against B-CLL, as well astherapeutic modalities useful against other lymphoproliferativedisorders.

One aspect of the invention relates to transgenic animals that expressTCL1 in B cells. In one embodiment of the invention, a transgenic animalis provided whose genome comprises a nucleic acid construct or transgenecomprising at least one transcriptional regulatory sequence capable ofdirecting expression to B cells operably linked to a nucleic acidsequence encoding TCL1. In a preferred embodiment of the invention, thetransgene comprises a DNA sequence encoding TCL1 which has been placedunder the transcriptional control of a V_(H) promoter and a Ig_(H)-μenhancer. In such animals, TCL1 expression is directed to immature andmature B cells. In one embodiment of the invention, the transgenicanimals are mice which develop an expanded population of B cells thatexpress the cell surface marker CD5. As the animals age, they developlymphocytic leukemia involving CD5+ B cells. This condition exhibitscharacteristics of human B-CLL.

In another embodiment of the invention, white blood cells from atransgenic animal exhibiting lymphoproliferation may be transferred to asecond animal (which may be a non-transgenic animal), thereby inducing arapid onset of lymphoproliferative disease in the second “recipient”animal.

According to another embodiment of the invention, potential therapeuticmodalities for preventing and/or treating lymphoproliferative disordersmay be tested by measuring the anti-lymphoproliferative activity of suchmodalities in animals produced according to one or more aspects of theinvention. Such activity may be assessed by measuring the capacity of apotential therapeutic modality to inhibit, prevent, and/or destroy oneor more of the symptoms or indications of lymphoproliferative diseaseexhibited by transgenic animals produced according to one embodiment ofthe invention and/or in “recipient” animals produced according toanother embodiment of the invention as described above. Such therapeuticmodalities, such as, for example, chemical compounds, will be formulatedin accordance with known methods to produce pharmaceutically acceptablecompositions. Such compositions may be administered to patients in avariety of standard ways.

EXAMPLES

The invention is further demonstrated in the following examples. All ofthe examples are actual examples. The examples are for purposes ofillustration and are not intended to limit the scope of the presentinvention.

Materials and Methods

Eμ-TCL1 Transgenic Mice

A 350-bp fragment possessing the entire human TCL1 coding region wasgenerated by PCR and cloned into the EcoRV and SalI sites of thepBSVE6BK (pEμ) plasmid containing a mouse VH promoter (V186.2) and theIgH-μ enhancer along with the 3′ untranslated region and the poly (A)site of the human β-globin gene. The construct containing TCL1 free fromvector sequences was injected into fertilized oocytes from B6C3 animals.Mice were screened for the presence of the transgene by Southern blotanalysis on tail DNAs digested with XhoI. Blots were hybridized with thesame BssHII DNA fragment used to inject the oocytes. Two founders wereobtained (F3 and F10) and bred. Transgenic heterozygote mice issued fromthese founders were studied and compared with nontransgenics siblingsraised in identical conditions. Genotyping was performed on tail DNAs byPolymerase chain reaction (PCR).

Western Blot Analysis

Cell proteins were extracted with NP-40 lysis buffer, quantified usingthe BCA kit (Pierce), size fractionated on 15% Tris-glycine SDS-PAGEgels and electrotransferred onto nitrocellulose (Immobilon-P,Millipore). The membrane was blocked overnight in 10% nonfat dried milkin PBST (phosphate-buffered saline (PBS): 7.6 g/L NaCl, 0.7 g/L Na2PO4,0.2 g/L KPO4 and 0.1% Tween 20). Expression was detected with the MoAb27D6/20 for human Tcl1 protein (14) according to ECL protocol(Amersham). Ponceau-S staining was employed to verify equivalent proteinloading.

Cell Preparations

Bone marrow cells were isolated by flushing the cavities of the femurand tibia with ice cold staining medium (Deficient RPMI, IrvineScientific) containing 10 mM HEPES, 0.1% NaN3, 3% FCS. Spleens weredissociated in staining medium between two frosted slides. Peritonealcells were removed by injection of 10 ml of staining medium into theperitoneal cavity following by withdrawal of the peritoneal exudates.Erythrocytes were lysed by brief treatment with 0.165 M ammoniumchloride and the cells then washed in staining medium.

White Blood Cell Preparation

Blood was collected from the cavernous sinus with a capillary tube in atube coated with EDTA (Becton Dickinson). Smears were immediatelyprepared and stained with May Grünwald Giemsa. Full counts were made ona cell counter (Beckman). For immunofluorescence staining cells weretreated with 0.165 M ammonium chloride to eliminate red cells and washedin staining medium.

Immunofluorescence Analysis and Cell Sorting

Single cell suspension of the indicated cell type were prepared andstained for surface expression as described previously (Hardy, R. R.,Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. (1991) J.Exp. Med. 173, 1213-1225). Cells were stained for surface expression ofIgM and CD5, then fixed and permeabilized using the Fix&Perm kit(CalTag) and stained for expression of Tcl1 using a PE-labeled anti-Tcl1monoclonal antibody 27D6/20 (Narducci, M. G., Pescarmona, E., Lazzeri,C., Signoretti, S., Lavinia, A. M., Remotti, D., Scala, E., Baroni, C.D., Stoppacciaro, A., Croce, C. M., et al. (2000) Cancer Res. 60,2095-2100). IgM/CD5 distributions were gated as indicated and histogramsof the Tcl1 staining determined. Plots were done with FlowJo software(Tree Star, Inc.). Exclusion of propidium iodide was used to eliminatedead cells and samples shown were also gated by forward and right anglescatter to exclude non-lymphoid cells and debris. Flow cytometry andsorting was done on a dual-laser dye-laser FACStarPLUS equipped withdetectors for 5 colors immunofluorescence. Samples were held on iceduring sorting. Preparation of labeled reagents has been describedpreviously (Hardy, R. R. (1986) in: The Handbook of ExperimentalImmunology 4^(th) Edition, eds. Weir, D. M., Herzenberg, L. A.,Blackwell, C. C. & Herzenberg L. A. (Blackwell scientific Pub. Ltd.,Edinburgh), pp. 31.1-31.12).

Analysis of VH11 Sequences

Cells were stained for IgM/CD5 expression and 1×10⁵ IgM+CD5+ cells weresorted directly into lysis/denaturation buffer. RNA and cDNA wereprepared as described previously (Li, Y. S., Wasserman, R., Hayakawa, K.& Hardy, R. R. (1996) Immunity 5, 527-535) and a VH11-Cμ fragment wasamplified using a VH11 leader and Cμ primer using Pfu polymerase andPCR. The amplified material was cloned using the TOPO TA cloning kit(Invitrogen) following manufacturer's instruction. Colonies with insertwere expanded, plasmid DNA isolated and sequenced using an ABI 377automated sequencer as described previously (Hardy, R. R., Carmack, C.E., Li, Y. S. & Hayakawa, K. (1994) Immunol. Rev. 137, 91-118).

Analysis of Cell Cycle

For Propidium Iodide (PI) staining, 10⁵ cells were sorted directly intoice cold 95% ethanol, nuclei were pelleted, then resuspended inPI-labeling solution (1 mg/ml RNAse A, 20 μg/ml propidium iodide in PBScontaining 0.0.1% NP40). After 30 minutes cells were analyzed for PIfluorescence on a FACScan using doublet discrimination gating.

Analysis of Cell Proliferation

Mice were injected intraperitoneally with 5-Bromodeoxyuridine (BrdU) inPBS at a dose of 50 μg per gram of body weight daily for four days. Micewere sacrificed and cells were stained for expression of IgM and CD5,then sorted to obtain the indicated populations. 5×105 cells were fixedand permeabilized, then treated with DNAse in acid buffer and stainedwith an anti-BrdU monoclonal antibody labeled with FITC. Samples wereanalyzed on a FACScan for BrdU staining.

Analysis of IgH Gene Rearrangement

Southern blots of DNA digested with EcoRI and StuI were preparedfollowing conventional methods and hybridized with a 32P-labeled DNAprobe PJ3 representing the JH4 region of the IgH locus.

The probe was synthesized by PCR amplification from mouse DNA withprimers F2 (5′-TGTGGTGACATTAGAACTGAAGTA-3′) (SEQ ID NO:1) and R1(5′-CAAGATTAGTCTGCAATGCTCAGA-3′) (SEQ ID NO:2).

Long Distance Inverse PCR (LDI-PCR)

High molecular weight DNA was digested with StuI. LDI-PCR was performedas described (Willis, T. G., Jadayel, D. M., Coignet, L. J. A.,Abdul-Rauf, M., Treleaven, J. G., Catovsky, D. & Dyer, M. J. S. (1997)Blood 90, 2456-2464). Primers designed within the mouse JH and IGHenhancer regions were used to amplify the purified DNA and thegel-purified products were ligated into pCR 2.1-TOPO vector(Invitrogen). Plasmids containing the correct size insert were sequencedusing an ABI 377 automated sequencer and compared with the Genbankdatabase using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).The VH, DH and JH segments were identified using the Genbank database.

Histopathology and Immunohistochemistry

Animals were autopsied and tissues were fixed in 10% buffered formalinand embedded in paraffin. Sections were stained with hematoxylin andeosin according to standard protocols and analyzed by mouse pathologists(University of Missouri, Research Animal Diagnostic Laboratory).Immunohistochemistry was performed on representative sections. For thedewaxing step the sections were heated for 1 h at 55° C. followed by arehydratation steps through a graded ethanol series and distilled water,immersed in PBS then treated with 0.1% trypsin solution in Tris bufferfor 30 min at 37° C. Endogenous peroxidase was blocked with 10% normalserum. The 27D6/20 MoAb specific for recombinant human Tcl1 protein (14)was used as a primary antibody and the immunohistochemical staining wasperformed by using streptoavidin-biotin peroxidase labeling methodaccording to the manufacturer's instructions (Histomouse-SP kit, Zymed).

Example 1 Production and Characterization of Eμ-TCL1 Transgenic Mice

Transgenic mice were generated in which the expression of TCL1 was underthe control of a VH promoter-IgH-Eμ enhancer whose activity specificallytargets expression of the transgene to immature and mature B-cells(Shaw, A. C., Swat, W., Ferrini, R., Davidson, L. & Alt, F. W. (1999) J.Exp. Med. 189, 123-129) (FIG. 1 a). Two transgenic founders on a B6C3background, designated F3 and F10, were generated and bred to establishtwo transgenic lines (FIG. 1 b). The expression of the transgene in eachwas evaluated by western blot of total protein extracted from spleen,bone marrow and liver of 3-month-old mice, using a monoclonal antibodyspecific for human Tcl1 protein (, M. G., Pescarmona, E., Lazzeri, C.,Signoretti, S., Lavinia, A. M., Remotti, D., Scala, E., Baroni, C. D.,Stoppacciaro, A., Croce, C. M., et al. (2000) Cancer Res. 60,2095-2100). The two transgenic lines expressed Tcl1 in spleen and bonemarrow while no expression was detected in liver or in non-transgenicsiblings (FIG. 1 c). Fluorescence-activated cell sorting (FACS) was alsoused to investigate the distribution of TCL1 expression on gated subsetsof B cells derived from spleen and peritoneal cavity of 3-month-oldmice, in both transgenic lines. The combination of cell surface markerswith intracellular detection of Tcl1 revealed a high level of TCL1expression in normal resting B cells with a 2.5-fold higher level in theCD5+ cells (FIG. 1 d).

Example 2 Phenotypic Analyses of Eμ-TCL1 Transgenic Mice

As demonstrated below, flow cytometric analysis revealed a markedlyexpanded CD5+ population in the peritoneal cavity of Eμ-TCL1 micestarting at two months of age that became evident in the spleen by 3-5months and in the bone marrow by 5-8 months. Analysis of immunoglobulingene rearrangements indicated monoclonality or oligoclonality in thesepopulations suggesting a preneoplastic expansion of CD5+ B cell clones.

The Immunophenotyping of Eμ-TCL1 Transgenic Mice Reveals an ExpandedCD5+/IgM+ Population

Flow cytometry was used to monitor the immunophenotypic profile ofperipheral blood lymphocytes (PBLs) from mice of these two lines betweenone and nine months of age. The results revealed the presence of aB220low/IgM+ population that was detected starting at six months of agein 100% of the transgenic mice, but in the absence of any sign ofdisease. A normal distribution of B cell populations was found in thenon-transgenic controls. T cell subsets were normal and identicalbetween transgenic animals and their littermate controls. Eμ-TCL1transgenic mice were further characterized in order to identify the Bcell subsets affected. The expanded B220low/IgM+ population was found toco-express CD5 and Mac-1/CD11b. This result suggested that the Eμ-TCL1transgenic mice had an expanded population of CD5+/B1 cells inperipheral blood, where such cells are normally infrequent (Kantor, A.B. & Herzenberg, L. A. (1993). Annu. Rev. Immunol. 11, 501-538). Inmice, CD5 is a pan-T cell surface marker that is also present on asubset of B-lymphocytes that appear during fetal/neonatal time, andwhose development appears quite distinct from the majority of B cells(Hardy, R. R., Carmack, C. E., Li, Y. S. & Hayakawa, K. (1994) Immunol.Rev. 137, 91-118; Hayakawa, K. & Hardy, R. R. (1988) Annu. Rev. Immunol.6, 197-218). CD5 is also frequently expressed on murine B cell lymphomasand leukemias (Lanier, L. L., Warner, N. L., Ledbetter, J. A. &Herzenberg, L. A. (1981) J. Exp. Med. 153, 998-1003; Phillips, J. A.,Mehta, K., Fernandez, C. & Raveché, E. S. (1992) Cancer Res. 52,437-443). A group of animals was analyzed at 2, 4 and 8 months of age toassess the expansion of the CD5+//IgM+ population in bone marrow, spleenand peritoneal cavity. FACS analysis revealed a phenotypicallyhomogeneous population markedly expanded in the peritoneal cavity of thetransgenic mice starting at two months of age (44%) that became evidentin spleen (8.6%) by 4 months and bone marrow by 8 months (43%) (FIG. 2a).

Histological and Immunocytochemical Analysis of the Transgenic Mice

Eight-month-old transgenic mice presented a slightly enlarged spleen,1.5-fold compared to littermate controls and moreover a very highcellularity in the peritoneal cavity, ranging between 50- to 100-foldincreased. Histopathology of enlarged spleens of Eμ-TCL1 micedemonstrated a consistent increase in the size of the marginal zone (MZ)(FIG. 2 b). Immunostaining of lymphoid cells in the white pulp of thespleen showed Tcl1 staining more intensely in the MZ. As expected noimmunostaining was observed in the spleen of littermate controls (FIG. 2c). Interestingly, the anatomical localization of the expanded CD5+cells was in the MZ whereas they did not have the precise phenotype oftypical MZ B cells, i.e. not CD21-high but rather CD21-low, like anormal CD5+ B cell (Chen, X., Martin, F., Forbush, K. A., Perlmutter, R.M. & Kearney, J. F. (1997) Int. Immunol. 9, 27-41). The histologicalanalysis of other tissues from the same animals, including thymus,liver, kidney and intestine, did not reveal any pathologic alteration(not shown).

Analysis of VH11 Sequences in the Expanded CD5+ Population

The increased frequency of CD5+ B cells in these transgenic mice couldrepresent either the induction of CD5 expression on cells normally notCD5+ or else the expansion of normally generated CD5+ B cells. In orderto distinguish between alternatives we investigated V gene usage in theexpanded cell population. Recurrent expression of certain VHVLcombinations is a characteristic feature of normal and neoplastic CD5+ Bcells (, R. R., Carmack, C. E., Li, Y. S. & Hayakawa, K. (1994) Immunol.Rev. 137, 91-118; Pennell, C. A., Arnold, L. W., Haughton, G. & Clarke,S. H. (1988) J. Immunol. 141, 2788-2796). Using antibodies specific forvariable regions, we found that one of these combinations, VH11Vk9, wasrepeatedly represented at 5-10% in the expanded CD5+ B cell populationin all mice analyzed, similar to the frequency seen in normal CD5+ Bcells (data not shown). Furthermore, analysis of VH11 sequences fromsorted IgM+/CD5+ cells from the spleen of a 3-month-old transgenic mouse(Table 1) showed normal VH11 rearrangements with low levels of N-regionaddition, typical of CD5+ B cells that are predominantly generatedfetally/neonatally when levels of TdT are low (Li, Y. S., Hayakawa, K. &Hardy, R. R. (1993) J. Exp. Med. 178, 951-960).

TABLE 1 Analysis of V_(H)11 sequences in CD5⁺ splenic B cells Seq. IDV_(H)11 N D_(H) N J_(H) D_(H) J_(H) TCL1-4 TGTATGAGA TA TAGTAGCTACTGGTACTTC DFL16.1 J_(H)1 (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO:11) TCL1-11 TGTATGAGA TATGGTAAC TACTGGTACTTC DSP2.8 J_(H)1 (SEQ ID NO:3) (SEQ ID NO: 5) (SEQ ID NO: 11) TCL1-17 TGTATGAGA TACGGTAGT AGCTACTGGTACTTC DFL16.1 J_(H)1 (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO:11) TCL1-42 TGTATGAGA TATGGTAAC TACTGGTACTTC DSP2.8 J_(H)1 (SEQ ID NO:3) (SEQ ID NO: 5) (SEQ ID NO: 11) TCL1-44 TGTATGAGA TATGGTAACTACTACTGGTACTTC DSP2.1 J_(H)1 (SEQ ID NO: 3) (SEQ ID NO: 7) (SEQ ID NO: 11)TCL1-46 TGTATGAGA TACGGTAGTAGC TACTGGTACTTC DFL16.1 J_(H)1 (SEQ ID NO:3) (SEQ ID NO: 8) (SEQ ID NO: 11) TCL1-47 TGTATGAGA TATGATGGTTACTACTGGTACTTC DSP2.9 J_(H)1 (SEQ ID NO: 3) (SEQ ID NO: 9) (SEQ ID NO: 11)TCL1-52 TGTATGAGA TATAGTAAC TACTGGTACTTC DSP2.X J_(H)1 (SEQ ID NO: 3)(SEQ ID NO: 10) (SEQ ID NO: 11)

IgM+/CD5+ Populations Are Arrested in the G0/G1 Phase of the Cell Cycleand Do Not Actively Divide

Chronic lymphocytic leukemia cells is characterized by a lowproliferative activity and by the progressive accumulation of clonal Blymphocytes blocked in the early phases (G0/G1) of the cell cycle(Andreeff, M., Darzynkiewicz, Z., Sharpless, T. K., Clarkson, B. D. &Melamed, M. R. (1980) Blood 55, 282-293; Nilsson, K. (1992) in ChronicLymphocytic Leukemia: Scientific Advances & Clinical development, ed.Cheson, B. D. (New York), pp. 33-45). The present inventor investigatedthe cell cycle distribution and the rate of cell proliferation in spleenand peritoneal cavity of four transgenic mice and four littermatecontrols at 7 months of age. Detection of DNA content in replicatingcells by propidium iodide (PI) labeling and analysis of cellproliferation from the distribution of 5-bromo-2′deoxyuridine (BrdU)incorporation in IgM+CD5+ sorted populations revealed that most of thesecells are not actively cycling in the transgenic mice (FIG. 2 d,e).

IgH Gene Configuration in Transgenic Mice with the Expanded CD5+Population

Analysis of Ig gene rearrangement revealed the presence of preleukemicor leukemic clones consistently in Eμ-TCL1 mice over seven months ofage. No clonality was observed in the youngest transgenics or innon-transgenic mice (FIG. 3 a). The detection of clonal JHrearrangements indicated that there could be a clonal expansion withoutevidence of disease. Further analysis of Ig gene rearrangement in bonemarrow, spleen and peritoneal cavity from 8-month-old mice with amarkedly expanded CD5+/IgM+ population showed an identical size JH banddetected in spleen and peritoneal cavity, but not bone marrow (FIG. 3b). The clonal JH band was not always shared between spleen andperitoneal cavity; note, for example, mouse # 40 (FIG. 3 b), which showstwo independent clonal populations, suggesting that multiple independentevents may occur in some cases. Clonal rearrangements were subsequentlyconfirmed in some samples by cloning and sequencing the rearranged bandusing a long-distance inverse polymerase chain reaction (LDI-PCR)(Willis, T. G., Jadayel, D. M., Coignet, L. J. A., Abdul-Rauf, M.,Treleaven, J. G., Catovsky, D. & Dyer, M. J. S. (1997) Blood 90,2456-2464). Using this approach, the transgenic mice exhibitedadditional clonal rearrangements compared to the littermate controls.Table 2 shows sequence data referring to the (+) samples marked as 9mand 8m in FIG. 3 a, StuI digested. Some sequences had a low level of Naddition, whereas others had a higher level (Table 2), as has been notedin sequence analyses of normal CD5+ B cells (Kantor, A. B., Merrill, C.E., Herzenberg, L. A. & Hillson, J. L. (1997) J. Immunol. 158,1175-1186). The clonal population suggested by Southern blot for thetransgenic # 41 (FIG. 3 b) in spleen and peritoneal cavity was alsoconfirmed by LDI-PCR (Table 2).

TABLE 2 Results of VDJ rearrangements in selected cases of Eμ-TCL1transgenic mice Mouse V_(H) D_(H) J_(H) V_(H) D_(H) J_(H) 9m TACTGTGCCAaATGGTTAC CTATGCTATGGACTACTG Vox-1 DSP2.6 J_(H)4 GA GA GGGTCAAGGAACCTCAG(SEQ ID NO: 12) (SEQ ID TCACCGTCTCCTCA NO: 15) (SEQ ID NO: 19) 9mTACTGTGCCA ACGGTAGT CTATGCTATGGACTACTG Vox-1 DFL16.1 J_(H)4 GA AGCcctGGGTCAAGGAACCTCAG (SEQ ID NO: 12) SEQ ID TCACCGTCTCCTCA NO: 16) (SEQ IDNO: 19) 8m GTCTATTACT actccccACTA CTGGTACTTCGATGTCTG V130 DFL16.1 J_(H)1GT CGGTAGTA GGGCACAGGGACCACGG (SEQ ID NO: 13) GCct TCACCGTCTCCTGA (SEQID (SEQ ID NO: 20) NO: 17) #41 GCAGGAGAC TATGGTTA CTGGTACTTCGATGTCTGNC1- DSP2.6 J_(H)1 PerC AGA (SEQ ID GGGCACAGGGACCACGG A7 (SEQ ID NO: 14)NO: 18) TCACCGTCTCCTCA (SEQ ID NO: 20) #41 GCAGGAGAC TATGGTTACTGGTAGTTCGATGTCTGGG NC1- DSP2.6 J_(H)1 Spleen AGA (SEQ IDGCACAGGGACCACGGTCAC A7 (SEQ ID NO: 14) NO: 18) CGTCTCCTCA (SEQ ID NO:20) Variations from the germline sequence are underlined. N regions arein lowercase.

Example 3 Eμ-TCL1 Mice Developed Lymphocytic Leukemia Upon Aging

Older mice eventually develop a CLL-like disorder resembling human B-CLLThe onset of a frank leukemia in the elderly mice provided furtherevidence of the establishment of a murine model for B-CLL. All micearound the age of 13-18 months became visibly ill and presented withenlarged spleens and livers associated with high white blood cell (WBC)counts. The weight of the transgenic spleens was between 1.5 g and 2.3 g(normal splenic weight was 0.07±0.01 g) and the mean of the WBC180.0×10⁶ cells/ml (the mean WBC/ml blood for normal adult mice was2.8×10⁶ cells/ml). In addition the mice also developed advancedlymphoadenopathy, a hallmark of human CLL. Cytological examination ofblood smears showed an increase in circulating lymphocytes with many ofthem displaying a clumped nuclear chromatin (FIG. 4 a,b). Thepredominant cell type was represented by large lymphoid cells andsmudged cells were also present. Histopathological examinationdemonstrated consistent infiltration of spleen, liver and lymph nodes bysmall and large lymphocytes (FIG. 4 c,e,g). Positive staining for Tcl1protein was observed primarily in lymphocytes found in these tissues(FIG. 4 d,f,h) and flow cytometric analysis confirmed the expansion ofthe CD5+/IgM+ population in all tissues (data not shown). Clonality wasshown by southern blot analysis of DNA isolated from leukemicsplenocytes using the PJ3 probe (FIG. 5). DNA from spleens of littermatecontrols showed the IgH gene in its germ-line configuration, whereas DNAfrom leukemic splenocytes presented extra-rearranged bands, indicatingthe presence of clonal B cell populations.

The above findings provide an animal model for CLL, the most commonhuman leukemia, and demonstrate that deregulation of the Tcl1 pathwayplays a crucial role in CLL pathogenesis. For additional discussion, seeRoberta Bichi, Susan A. Shinton, Eric S. Martin, Anatoliy Koval, GeorgeA. Calin, Rossano Cesari, Giandomenico Russo, Richard R. Hardy and CarloM. Croce, Human chronic lymphocytic leukemia modeled in mouse bytargeted TCL1 expression, (2002) Proc. Natl. Acad. Sci. USA 99 (10):6955-6960, the disclosure of which is hereby incorporated herein byreference in its entirety.

Example 4

The lymphoproliferative condition exhibited by transgenic animalsaccording to an embodiment of the invention was found to betransplantable to syngeneic animals.

Method to Expand Primary CLL Lymphomas

Moribund Eμ-TCL1 transgenic mice were sacrificed and autopsied. Spleenor, less frequently, lymph node white cells were isolated, counted,diluted in PBS and injected IP in syngeneic mice at 100, 10, 1 million,100 or 10 thousand cells per mouse. A few cells from affected tissueswere stained for IgM, B220, and/or CD5 for FACS analysis. Bone marrowcells were collected for cytogenetics.

TABLE 3 Effects of primary CLL tumors injected in syngeneic mice #91(founder # 7092) injected Jun. 7, 2002 (10.2 months ago) all males with100 millions cells died all females with 100 millions cells died allmales with 10 millions cells died all females with 10 millions cellsdied all males with 1 millions cells died all females with 1 millionscells died 4/5 males with 100 thousand cells died 3/5 females with 100thousand cells died 3/5 males with 10 thousand cells died 0/5 femaleswith 10 thousand cells died tumor features: 1) males may be slightlymore affected than females; 2) mortality is cellular dose dependent; 3)homing unknown. #152 (founder # 7092) injected Aug. 28, 2002 (7.5 monthsago) all males with 100 millions cells died (surv. = 94) all femaleswith 100 millions cells are still alive 4/5 males with 10 millions cellsdied all females with 10 millions cells are still alive 3/5 males with 1millions cells died 4/5 females with 1 millions cells are still aliveall males with 100 thousand cells died (surv. = 124) all females with100 thousand cells are still alive 3/5 males with 10 thousand cells diedall females with 10 thousand cells are still alive tumor features: 1)strong sexual dimorphism (only males are affected); 2) mortality isindependent of the amount of cells injected; 3) spleen and sometimesliver are the most affected tissues (enlarged lymph nodes were neverfound). #180 (founder # 7323) injected Oct. 4, 2002 (6.3 months ago) allmales with 100 millions cells died (surv. = 94) all females with 100millions cells died (surv. = 84) all males with 10 millions cells died(surv. = 107) 4/5 females with 10 millions cells died all males with 1millions cells died (surv. = 123) 3/5 females with 1 millions cells died4/5 males with 100 thousand cells died 1/5 females with 100 thousandcells died 4/5 males with 10 thousand cells died 0/5 females with 10thousand cells died tumor features: 1) males are more affected thanfemales; 2) mortality is dependent on the amount of cells injected; 3)neck lymph nodes are the most affected tissues. #178 (founder # 7323)injected Nov. 26, 2002 (4.5 months ago) all males with 10 millions cellsdied (surv. = 88) all females with 10 millions cells died (surv. = 78)1/5 males with 1 millions cells died 3/5 females with 1 millions cellsdied tumor features: 1) females seem to be more affected than males; 2)mortality is dependent on the amount of cells injected; 3) formation ofa large amount of ascitic fluid; thymic and mesenteric lymph nodes arethe most affected tissues. #232 (founder # 7323) injected Dec. 19, 2002(3.8 months ago) 3/5 males with 10 millions cells died 0/5 females with10 millions cells died 1/5 males with 1 millions cells died 0/5 femaleswith 1 millions cells died tumor features: 1) males seem to be moreaffected than females; 2) mortality is dependent on the amount of cellsinjected; 3) tumor phenotype not yet studied. #243 (founder # 7092)injected Jan. 9, 2003 (3.2 months ago) all mice are alive + 2 moretumors injected 2 months ago

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

1. A transgenic mouse whose genome comprises a nucleic acid sequenceencoding a human TCL1, wherein said sequence is operably linked to aV_(H) promoter and to a IgH-μ enhancer, wherein said transgenic mousedevelops an expanded population of CD5+ B cells compared to a controlmouse.
 2. The transgenic mouse of claim 1, wherein said V_(H) promotercomprises a mouse V_(H) promoter.
 3. The transgenic mouse of claim 1,wherein said IgH-μ enhancer comprises a mouse IgH-μ enhancer.
 4. Thetransgenic mouse of claim 1, wherein said mouse develops a lymphocyticleukemia which exhibits characteristics of human B-CLL.
 5. A transgenicmouse whose genome comprises a nucleic acid sequence encoding a humanTCL1, wherein said sequence is operably linked to a V_(H) promoter andto a IgH-μ enhancer, and wherein said transgenic mouse develops alymphocytic leukemia that exhibits characteristics of human B-CLL. 6.The transgenic mouse of claim 5, wherein said V_(H) promoter comprises amouse V_(H) promoter.
 7. The transgenic mouse of claim 5, wherein saidIgH-μ enhancer comprises a mouse IgH-μ enhancer.